Although several techniques are currently available for studying DNA methylation, each has significant limitations. This proposal describes the development of a Methylation Ratio Assay (MRA) that specifically collects hypermethylated DNA and hypomethylated DNA in parallel reactions, using affinity pull-downs with the methyl-DNA binding domain containing heterodimer MBD2/MBD3L 1, and the CXXC domain of MBD1 a respectively! and allows determination of the methylation ratio for individual loci. We intend to optimize expression and storage of these protein domains, and develop the protocols to capture their respective target DNAs using templates containing 1 to 16 CpGs, and subsequently genomic DNA-either non-methylated or in vitro methylated with M.Sssl. Final assay validation will include survey of 10 to 20 loci in the breast cancer cell lines MCF-7 and MDA-MB-468. Active Motif specializes in kit production, and will launch this kit within our existing infrastructure. Its future potential for early cancer diagnostic method will be further evaluated through collaborators with access to clinical samples. Changes in DNA methylation patterns is a characteristic commonly found in many diseases including breast cancer. This proposal describes a novel assay that enables detection of both increases and decreases in DNA methylation whose development may enable early diagnosis.